Aseptic tissue processing method, kit and device

ABSTRACT

The present invention concerns a single use aseptic kit comprising: a disaggregation module for receipt and processing of material comprising solid mammalian tissue; and a stabilisation module for storing disaggregated product material, wherein each of the modules comprises one or more flexible containers connected by one or more conduits adapted to enable flow of the tissue material there between; and wherein each of the modules comprises one or more ports to permit aseptic input of media and/or reagents into the one or more flexible containers. The invention further relates to an automated device for semi-automated aseptic disaggregation and/or enrichment and/or stabilisation of cells or cell aggregates from mammalian solid tissue comprising a programmable processor and the single use aseptic kit. The invention further relates to a semi-automatic aseptic tissue processing method.

The present invention concerns a kit and a semi-automatic device usingthat kit for aseptic disaggregation of solid tissue derived eukaryoticcells into either single cells or small cell number aggregates. Theinvention further relates to a semi-automatic aseptic tissue processingmethod comprising: a process for aseptic disaggregation of solid tissuederived eukaryotic cells into either single cells or small cell numberaggregates and their further processing.

BACKGROUND

The conditions during solid tissue disaggregation and time taken toharvest the cells have a substantial impact on the viability andrecovery of the final cellularised material. Typically a solid tissuederived cell suspension, that is obtained, comprises a wide variety ofdifferent cell types and the disaggregation media and tissue debris orfluids. Often, selective targeting and or isolation of an individual ormultiple cell types is prerequisite for the starting material prior tomanufacture of regenerative medicines, adoptive cell therapies, ATMPs,diagnostic in vitro studies or scientific research. Generally theseselection or enrichment techniques rely on one of the followingproperties: size, shape, density, adherence or strong protein: proteininteractions (i.e. antibody: antigen) or providing a growth supportingenvironment by controlling the culture conditions or more complex cellmarker interactions associated with semi-permanent or permanent couplingto magnetic or non-magnetic solid or semi-solid phase substrates can beused.

For enrichment, isolation or selection in principle any sortingtechnology can be used. This includes for example affinitychromatography or any other antibody-dependent separation techniqueknown in the art. Any ligand-dependent separation technique known in theart may be used in conjunction with both positive and negativeseparation techniques that rely on the physical properties of the cells.An especially potent sorting technology is magnetic cell sorting.Methods to separate cells magnetically are commercially available e.g.from Thermo fisher, Miltenyi Biotech, Stem cell Technologies, Cellpro,Seattle, Advanced Magnetics, Boston or Quad Technologies Boston. Forexample, monoclonal antibodies can be directly coupled to magneticpolystyrene particles like Dynal M 450 or similar magnetic particles andused e.g. for cell separation. The Dynabeads technology is not columnbased, instead these magnetic beads with attached cells enjoy liquidphase kinetics in a sample tube, and the cells are isolated by placingthe tube on a magnetic rack. However, in a preferred embodiment forenriching, sorting and/or detecting neuronal cells from a samplecontaining neuronal cells according the present invention monoclonalantibodies are used in conjunction with colloidal superparamagneticmicroparticles having an organic coating by e.g. polysaccharides(Magnetic-activated cell sorting (MACS) technology (Miltenyi Biotec,Bergisch Gladbach, Germany)). These particles (nanobeads or MicroBeads)can be either directly conjugated to monoclonal antibodies or used incombination with anti-immunoglobulin, avidin or antihapten-specificMicroBeads or coated with other mammalian molecules with selectivebinding properties.

Magnetic particle selection technologies such as those described above,allows cells to be positively or negatively separated by incubating themwith magnetic nanoparticles coated with antibodies or other moietiesdirected against a particular surface marker. This causes the cellsexpressing this marker to attach to the magnetic nanoparticles.Afterwards the cell solution is placed within a solid or flexiblecontainer in a strong magnetic field. In this step, the cells attach tothe nanoparticles (expressing the marker) and stay on the column, whileother cells (not expressing the marker) flow through. With this method,the cells can be separated positively or negatively with respect to theparticular marker(s).

In case of a positive selection the cells expressing the marker(s) ofinterest, which attached to the magnetic column, are washed out to aseparate vessel, after removing the column from the magnetic field.

In case of a negative selection the antibody or selective moiety used isdirected against surface markers(s) which are known to be present oncells that are not of interest. After application of the cells/magneticnanoparticles solution onto the column the cells expressing theseantigens bind to the column and the fraction that goes through iscollected, as it contains the cells of interest. As these cells arenon-labelled by the selective antibodies or moiety(s) coupled tonanoparticels, they are “untouched”.

The known manual or semi-automated solid tissue processing steps arelabour-intensive and require a knowledge of the art.

In addition where the material is used for therapeutic purposes, theprocessing requires strict regulated environmental conditions duringhandling the cell cultures, for example tissue processing as apart of orprior to disaggregation; enzymatic digestion and transfer into storingdevices or incubation conditions for disaggregation/cellularisation andviable tissue yields. Typically this process would require multiplepieces of laboratory and tissue processing equipment, and personal withthe skills and knowledge of the scientific art with critical stagescontained within either hazard containment or tissue processingfacility(s) aseptic environment(s) in order to perform the same activitysafely and also minimise the risk of contamination(s).

The invention therefore arises from a need to provide improved solidtissue processing, including an apparatus/device that undertakes saidprocessing that achieves the unmet need described above.

SUMMARY OF INVENTION

The present invention concerns a single use aseptic kit comprising adisaggregation module for receipt and processing of material comprisingsolid mammalian tissue; an optional enrichment module for filtration ofdisaggregated solid tissue material and segregation of non-disaggregatedtissue and filtrate; and a stabilisation module for optionally furtherprocessing and/or storing disaggregated product material, wherein eachof said modules comprises one or more flexible containers connected byone or more conduits adapted to enable flow of the tissue material therebetween; and wherein each of said modules comprises one or more ports topermit aseptic input of media and/or reagents into the one or moreflexible containers.

In prior art the tissue may undergo physical and or enzymaticdisaggregation/cellularisation in a single container. In the presentinvention sets of containers which are interconnected and have specificseparate functions maintain an aseptically closed system to process,optionally enrich but stabilise the disaggregated and cellularised solidtissue product. Essentially the invention provides a rapidpre-sterilised environment to minimise the time required and risk ofcontamination or operator exposure during the processing of the solidtissue.

The kit described here allows for closed solid tissue processingeliminating the risk of contamination of the final cellularised productcompared to standard non-closed tissue processing. This is especiallywhen the process is performed within a tissue retrieval/procurement siteand requires storage prior to final cell processing for its ultimateutility. In addition, safety of the operator is increased due toreduction of direct contact with biological hazardous material which maycontain infectious organisms such as viruses.

The kit also enables either all of or a portion of the finally processedcellularised material to be stabilised for either transport or storageprior to being processed for its ultimate utility.

The invention will enable the solid tissue to be processed at the timeof tissue retrieval, or later if required, without impact upon theretrieval procedure or the viability of the cellularised product.

In some embodiments employing optional enrichment via a form of physicalpurification to reduce impurities such as no longer required reagents;cell debris; non-disaggregated tissue and fats. A single cell or smallcell number aggregates can be enriched for stabilisation afterdisaggregation by excluding particles and fluids of less than 5 μm orincompletely disaggregated material of or around 200 μm across or largerbut this will vary upon the tissue and the efficiency of disaggregationand various embodiments in the form of tissue specific kits may beemployed depending upon the tissue or ultimate utility of thedisaggregated solid tissue.

In some embodiments the one or more flexible containers comprise aresilient deformable material. The one or more flexible containers ofthe disaggregation module may comprise one or more sealable openings.The one or more flexible containers of the disaggregation module and/orthe stabilisation module may also comprise a heat sealable weld.

In further embodiments the one or more flexible containers that are partof any module comprise internally rounded edges.

The one or more flexible containers of the disaggregation module maycomprise disaggregation surfaces adapted to mechanically crush and shearthe solid tissue therein.

Further, the one or more flexible containers of the enrichment modulemay comprise a filter adapted to retain a retentate of cellulariseddisaggregated solid tissue.

In embodiments, one or more flexible containers of the stabilisationmodule comprise media formulation for storage of viable cells insolution or in a cryopreserved state. In some embodiments the

In further embodiments the kit further comprises a radio frequency orother digitally recognisable identification tag so that it may bescanned and recognised during automated processing, such as with/in theautomated device in embodiments of the present invention. Crucially thetag provides information about the conditions and steps required to beauto processed, so simply by scanning the kit, any automated system usedwith the kit to process the tissue can be undertaken without furtherintervention or contamination. Once the tissue sample has been placed inthe disaggregation module, it can for example be sealed, manually, orautomatically, before processing begins.

In this regard, in preferred embodiments that include a device, the kitassociated tag is detected by the device's processor and the device thenruns a specific program according to a type of disaggregation and/orenrichment and/or stabilisation process; one or more types of media usedin those processes; including an optional freezing solution suitable forcontrolled rate freezing. Put another way, the kit is therefore bereadable by an automated device used to execute a specific fullyautomatic method for processing the specific tissue when inserted tosuch a device.

The invention is particularly useful in a sample processing,particularly automated processing. Thus, in a further aspect theinvention concerns use of the single use aseptic kit described above ina semi-automated process for the aseptic disaggregation and/orenrichment and stabilisation of mammalian cells or cell aggregates.

A particular advantage is that solid tissue disaggregation (and optionalprocesses including all described manipulations herein describedrequired to achieve optimal results) can be performed in a closedsystem, i.e. an aseptic process with minimal risk of contaminations andwith minimal user knowledge.

The invention further relates to an automated device for semi-automatedaseptic disaggregation and/or enrichment and stabilisation of cells orcell aggregates from mammalian solid tissue comprising a programmableprocessor and the single use aseptic kit as described in any of thebefore mentioned examples above.

In embodiments, as previously described, the device may have acomprising radio frequency identification tag reader to recognise thesingle use kit. The programmable processor is capable of recognising thesingle use aseptic kit via its tag and subsequently able to execute thekit programme which defines the type of disaggregation, enrichment andstabilisation processes together with the respective media typesrequired for those processes.

In this regard, the programmable processor is adapted to communicatewith and control one or more of: the disaggregation module; theenrichment module; and the stabilisation module of the device. Thedevice, including its processor, may therefore have multiplefunctionality to assess the flow of materials through the kit makingdecisions of when a step is completed as part of the pre-programmedfunctions and the feedback the device gets from its sensors.

For example, the programmable processor may control the disaggregationmodule to enable a physical and/or biological breakdown of the solidtissue material in that container. The programmable processor may alsocontrol the disaggregation module to enable a physical and enzymaticbreakdown of the solid tissue material.

In some embodiments the enzymatic breakdown of the solid tissue materialis by the selection and provision of one or more media enzyme solutionsselected from collagenase, trypsin, lipase, hyaluronidase,deoxyribonuclease, Liberase H1, pepsin, or mixtures thereof.

In addition or alternatively the programmable processor may controldisaggregation enabling the surfaces within the disaggregation flexiblecontainers to mechanically crush and shear the solid tissue. Inembodiments, the disaggregation surfaces are controlled by mechanicalpistons, for example.

In some embodiments, the programmable processor controls thestabilisation module to cryopreserve the enriched disaggregated solidtissue in the container, for example, this may be achieved by using aprogrammable temperature setting, a condition which is determined byreading the tag of the kit inserted in the device.

In some embodiments, to undertake different functions of the process,one or more of the additional components of the device and/or kit areprovided. Such features may be available in any combination. This mayinclude for example: sensors in the device capable of recognisingwhether a disaggregation process has been completed in thedisaggregation module of the kit prior to transfer of the disaggregatedsolid tissue to the optional enrichment module; weight sensors todetermine an amount of media required in the containers of one or moreof the disaggregation module; the enrichment module; and/or thestabilisation module and means to control that transfer of materialbetween respective containers; and temperature sensors to control thetemperature within the containers of the one or more of thedisaggregation module; the enrichment module; and/or the stabilisationmodule.

Other possible features include an optional bubble sensor to control thetransfer of media between the input and output ports of each containerin the module; one or more pumps may provide means to control thetransfer of media between the input and output ports; and/or pressuresensors to assess the pressure within the enrichment module; valves tocontrol an optional tangential flow filtration process within theenrichment module; and/or one or more clamps to control the transfer ofmedia between the input and output ports of each module.

For example, the programmable processor is adapted to maintain anoptimal storage temperature range in the stabilisation module until thecontainer is removed; or executes a controlled rate of freezing.

These embodiments of the device and kit allow solid tissue derived cellsor cell aggregates to be: stored for short periods (minutes to days) orstored for long periods (multiple days to years) prior to their ultimateutility depending on the type or stabilisation process used with thestabilisation module.

For ease of use, the device of the invention may further comprise a userinterface. That interface may comprise a display screen to displayinstructions that guide a user to input parameters, confirmpre-programmed steps, warn of errors, or combinations thereof.

In many cases it is desirable that the automated device is adapted to betransportable and thus may comprise dimensions that permit easymanoeuvrability and/or aid movement such as wheels, tyres, handles andthe like.

The final cellular material product can then be used for but not limitedto either: regenerative medicine, adoptive cell therapies, ATMPs,diagnostics or to further the basic scientific understanding of tissue,cell function or organism function.

The combination of an aseptic kit, automated processing device andassociated media formulations, which can disaggregate solid tissues toprovide functional living cells or the product of the cells forsubsequent therapeutic, diagnostic or scientific use, is thereforehighly desirable.

In some embodiments the cells produced using the kit and/or device ofthe invention are useful for providing functional living cells and maybecultured further for that use. Cell culture is a process by which cellsare grown outside the original host using controlled environmental andsupportive conditions which vary by cell type and organism. These areoften sterile artificial vessels which allow gas and temperature to bemaintained and either manual or automated changes in essentialnutrients, metabolites, growth factors and gases which enable regulationof the cells requirements to survive and in most cases thrive. Cellculture requirements differ broadly by the type of cell(s) and itsrequired purpose. Cell culture conditions can be optimised for cellexpansion, cell differentiation or manufacturing of different phenotypesof the cell or its products. The most commonly varied factor in culturesystems is the cell culture medium, for which a vast number of recipesis known (see for example “Cell Culture Techniques” Humana Press, 1st.Edition, 2011)

In some embodiments disaggregated or cellularised material produced bythe device and kit can be useful as the starting material to isolatespecific cell populations which are grown out using stimulation ornon-required cells are inhibited or apoptosis/cell death is inducedresulting in a semi/purified population.

Such cells can be further sorted by one or more of the followingprocesses: Fluorescence-activated cell sorting using antibody/proteinlabelling or natural fluorescence; Magnetic separation of cells, e.g.the magnetic activated cell sorting (MACS technology, Miltenyi BiotecGmbH, Germany). This technology requires a marker that allows directseparation of the cells of interest by an antibody coupled to a magneticmicrobead (Miltenyi et al., Cytometry 1990; 11:231-238). Alternatively,where it is not possible or not desirable to actively select the targetpopulation a process of negative isolation can be employed. In thisapproach, non-target cells are magnetically labelled and depleted,thereby leaving the unlabelled cells of interest; Label free cellseparation and sorting using physical separation methods where eitherthe target is not known, is a mixed population and physical cell (orclumps of cells) characteristics can be used to separate the cellularmaterial from the current media to: remove impurities or reagents thatare no longer required such as enzymes, cell debris, connective tissue,fat & mineral deposits; or exchange fluids which may be better forstabilising the cells for distribution and/or storage. It is envisagedthat embodiments of the invention may include such functionality withinthe parameters of the processor or the automated device and operatingsystem.

For example, the purity, of the disaggregated and cellularised solidtissue product, can be further increased if one or more cell surfacemarker(s) are used to select for or deplete a subpopulation of cellseither as an independent step within the process or after processingusing the methods described.

The present invention also relates to a method for enhancedsemi-automated disaggregation cellularisation and storage of tissuederived cells. Optionally, steps of enrichment, formulation andcryopreservation are also provided.

In a further aspect of the invention, there is provided a semi-automaticaseptic tissue processing method comprising: automatically determiningaseptic disaggregation tissue processing steps and one or more furthertissue processing steps and their associated conditions from a digitaltag identifier on an aseptic processing kit, optionally in accordancewith the kit described herein; placing a tissue sample into a flexibleplastic container of the aseptic processing kit; and processing thetissue sample by automatically executing the one or more tissueprocessing steps by communicating with and controlling thedisaggregation module; the optional enrichment module; and thestabilisation module.

The one or more automatically executed processes may be selected fromone or more of:

1) transferring media, preferably enzyme media, into the disaggregationchamber (for example, in accordance with the sealable disaggregationflexible container of the kit of the invention). The media maybetransferred into the disaggregation chamber, or in one embodiment alsoenters and collects enzymes prior to disaggregation using one or moreembodiments of the invention, e.g. a mechanism such as weight sensorswhich will assess the required amount of media to add either determinedby: direct operator input or weight of solid tissue. Incubating with themedia at an optimal temperature of between 30 & 37° C. but could be aslow as 0° C. upto 40° C. for at least 1 minute to several hours but morepreferable 15 to 45 minutes.

2) undertaking physical disaggregation for a minimum of a few seconds upto several hours with an optimal time of between 1 and 10 minutesrequired to break up the solid tissue until there is no visual change(Table 1). The disaggregation is designed to compress the tissues usinga variable speed and time depending upon the time taken to disaggregateand feedback via sensors within the disaggregation module.

Steps 1) or 2) may be repeated until the tissue stops changing or hasbeen disaggregated into a liquid cell suspension (whichever comes 1stmonitored by a sensor in the disaggregation module).

3) removing disaggregated tissues, associated material and impurities bypassing the disaggregated tissue and media through one or more filtersenabling optional enrichment of the cell suspension. Direct pass throughone or more mechanical filters with holes at least >0.1 μm to 1000 μmbut most preferably between 50 and 250 μm and more preferably 100 μm to200 μm. Alternatively, other separation methods maybe used such as:

-   -   I density based separation using centrifugation and/or        sedimentation with or without a cell aligned density retention        solution (e.g. Ficoll-paque GE Healthcare).    -   II Hydrodynamic filtration where fluid flow and flow obstructing        materials enhance the resolution and fractionation of the cells        and impurities based on size and shape    -   III. Field flow fractionation where an applied field (e.g. flow,        electric, gravitational, centrifugal) acts in a perpendicular or        reverse direction to the selection flow (e.g. Tangential flow        filtration, Hollow fibre flow filtration, Asymmetric flow        filtration, Centrifugal flow filtration). In which case: cells        or impurities which are most responsive to the force are driven        to the wall where flow is lowest and therefore a long retention        time; while cells or impurities which are least responsive to        the force remain laminar to the flow and elute quickly        (tangential flow filtration)    -   IV Acoustophoresis where one or more an acoustic frequency(ies)        tuned to or harmonized with populations of cells or impurities        is used to drive the required cells or impurities in a        tangential path to the input stream.

4. Re-suspending the disaggregated cell product in fresh or additionalmedia. This could be a cell enrichment media in order to undergo anindependent targeted enrichment procedure or direct cell culture or coldstorage media (such as HypoThermosol® from BioLife Solutions).

5. Transferring to a stabilising module containers for storage for hoursto days or

6. Re-suspending in, or addition of a, cryoprotectant—a freezingsolution for storage of the disaggregated solid tissue derived productfor days to years (such as CryoStor® Freezing solution from BioLifeSolutions) and transferring to one or more flexible stabilising modulehaving a cryopreservation container(s)

7. Performing a controlled rate freezing process

8. Separating the aseptic processing kit from the device for independentstorage or distribution.

During such steps it is apparent that the disaggregated module and thestorage module may comprise one and the same flexible container, forreceiving the sample and storing the sample and a further flexiblecontainer for housing the media for disaggregation. In some embodimentsthe same flexible containers are part of different modules of the kit.

DESCRIPTION OF THE INVENTION

The processing of tissue to cells according to the kit, semi-automateddevice and methods of present disclosure are described further in theaccompanying examples and figures numbered 1 to 7.

Moreover, by utilising the kit, device and processes described herein,in conjunction with ordinary skills in the art, further embodiments ofthe present disclosure can be readily identified. Those skilled in theart will readily understand known variations.

Definitions of the Disclosure

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

“depletion” as used herein refers to a process of a negative selectionthat separates the desired cells from the undesired cells which arelabelled by one marker-binding fragment coupled to a solid phase.

“disaggregation or disaggregate” as used herein refers to thetransformation of solid tissue into a single cells or small cell numberaggregates where a single cell as a spheroid has a diameter in the rangeof 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60μm, 70 μm, 80 μm, 90 μm, 100 μm or more where this is more usuallybetween 7 to 20 μm.

“cellularised or cellularisation” as used herein refers to the processof disaggregation where by the solid tissue a multicellular materialgenerally made up of multiple cell lineages/types is broken down intosmall numbers of cells including but not limited to one cell but couldbe multiple cells of various lineages or cell types in very smallnumbers i.e. clump of cells or cell aggregates.

“engineered” as used herein refers to either addition of nucleicmaterial or factors which change the tissue derived cell function fromtheir original function to have a new or improved function for itsultimate utility.

“filtrate” as used herein refers to the material that passes through afilter, mesh or membrane.

“flexible container” as used herein refers to a flexible packagingsystem in multiple formats with one or more different types of film.Each film type is selected to provide specific characteristics topreserve the physical, chemical, and functional characteristics of thesterile fluids, solid tissue derived cellular material and the containerintegrity depending upon the step of the process.

“freezing solution” or “cryopreservation solution” also referred in thefield to as the cryoprotectant is a solution that containscryoprotective additives. These are generally permeable, non-toxiccompounds which modify the physical stresses cells are exposed to duringfreezing in order to minimise freeze damage (i.e. due to ice formation).Most commonly a % Vol/Vol of one or more of the following:Dimethylsulphoxide (DMSO); Ethylene glycol; Glycerol;2-Methyl-2,4-pentanediol (MPD); Propylene glycol; Sucrose; & Treha lose.

“media” means various solutions known in the art of cell culturing, cellhandling and stabilisation used to reduce cell death, including but notlimited to one or more of the following media Organ PreservationSolutions, selective lysis solutions, PBS, DMEM, HBSS, DPBS, RPMI,Iscove's medium, X-VIVO™, Lactated Ringer's solution, Ringer's acetate,saline, PLASMALYTE™ solution, crystalloid solutions and IV fluids,colloid solutions and IV fluids, five percent dextrose in water (D5W),Hartmann's Solution. The media can be standard cell media like the abovementioned media or special media for e.g. primary human cell culture(e.g. for endothelia cells, hepatocytes or keratinocytes) or stem cells(e.g. dendritic cell maturation, hematopoietic expansion, keratonocytes,mesenchymal stem cells or T cell expansion). The media may havesupplements or reagents well known in the art, e.g. albumins andtransport proteins, amino acids and vitamins, antibiotics, attachmentsfactors, growth factors and cytokines, hormones, metabolic inhibitors orsolubilising agents. Various media are commercially available e. g. fromThermoFisher Scientific or Sigma-Aldrich.

“non-labelled” or “untouched” as used herein refers to the cells whichare not bound by one marker-binding fragment coupled to a solid phase.The non-labelled, untouched cell fraction contains the desired targetcells.

“non-target cells” as used herein refers to cells which are specificallybound by one marker-binding fragment which is coupled to a solid phasethat is used to remove an unwanted cell type.

“positively separated” as used herein refers to the active separation ofcells which are bound by one marker-binding fragment coupled to a solidphase and these cells are the required population of cells.

“negatively separated” as used herein refers to the active separation ofcells which are bound by one marker-binding fragment coupled to a solidphase and these cells are not the required population of cells.

“purity” as used herein refers to the percentage of the targetpopulation or populations desired from the original solid tissue.

“regenerative medicine(s)”, “adoptive cell therapy(ies)” or “advancedtherapy medicinal product(s)” are used interchangeably herein to referto cellular material that is used for therapeutic purposes of one ormore mammals either by: the action of a part of or all of the cellularmaterial; the supportive actions of a part of or all of the cellularmaterial with the aim to improve the wellbeing of the mammal afterapplication. The therapeutic cells can either be used directly or mayrequire further processing, expansion and/or engineering to providethese actions.

“sample” as used herein refers to a sample containing cells in anyratio. Preferentially, these cells are viable. But, these cells can alsobe fixed or frozen cells which may be used for subsequent nucleic acidsor protein extraction. The samples may be from animals, especiallymammals such as mouse, rats or humans. Any compressible solid tissuethat contains cells can be used. The invention is illustrated mainlythrough the isolation of hematopoietic and cancer cells from solidtumour tissue. However, the invention relates to a method for isolationof a breadth of cells from any mammalian solid tissue.

“marker” as used herein refers to a cell antigen that is specificallyexpressed by a certain cell type. Preferentially, the marker is a cellsurface marker, so that enrichment, isolation and/or detection of livingcells can be performed.

“solid phase” as used herein refers to the coupling of themarker-binding fragment, e.g. an antibody, bound to anothersubstrate(s), e.g. particles, fluorophores, haptens like biotin,polymers, or larger surfaces such as culture dishes andmicrotiterplates. In some cases the coupling results in directimmobilization of the antigen-binding fragment, e.g. if theantigen-binding fragment is coupled to a larger surface of a culturedish. In other cases this coupling results in indirect immobilisation,e.g. an antigen-binding fragment coupled directly or indirectly (viae.g. biotin) to a magnetic bead is immobilised if said bead is retainedin a magnetic field. In further cases the coupling of theantigen-binding fragment to other molecules results not in a direct orindirect immobilization but allows for enrichment, separation,isolation, and detection of cells according to the present invention,e.g. if the marker-binding fragment is coupled to a chemical or physicalmoiety which then allows discrimination of labelled cells andnon-labelled cells, e.g. via flow cytometry methods, like FACSsorting,or fluorescence microscopy.

“solid tissue” as used herein refers to a piece or pieces of animalderived mammalian solid tissue which by its three dimensions i.e.length, breadth and thickness as a geometrical body is larger than thesize of multiple individual cell based units and often containsconnective materials such as collagen or a similar matrix that make upstructure of the tissue whereby said solid tissue cannot flow throughtubes or be collected by a syringe or similar small conduit orreceptacle and is i.e. with dimensions in the range of 500 μm, 1 mm, 2mm, 3 mm, 4 mm, 5 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 10 cm, 20 cm, 30 cmor more

“particle” as used herein refers to a solid phase such as colloidalparticles, microspheres, nanoparticles, or beads. Methods for generationof such particles are well known in the field of the art. The particlesmay be magnetic particles or have other selective properties. Theparticles may be in a solution or suspension or they may be in alyophilised state prior to use in the present invention. The lyophilizedparticle is then reconstituted in convenient buffer before contactingthe sample to be processed regarding the present invention.

“magnetic” in “magnetic particle” as used herein refers to all subtypesof magnetic particles which can be prepared with methods well known tothe skilled person in the art, especially ferromagnetic particles,superparamagnetic particles and paramagnetic particles. “Ferromagnetic”materials are strongly susceptible to magnetic fields and are capable ofretaining magnetic properties when the field is removed. “Paramagnetic”materials have only a weak magnetic susceptibility and when the field isremoved quickly lose their weak magnetism. “Superparamagnetic” materialsare highly magnetically susceptible, i.e. they become strongly magneticwhen placed in a magnetic field, but, like paramagnetic materials,rapidly lose their magnetism.

“marker-binding fragment” as used herein refers to any moiety that bindspreferentially to the desired target molecule of the cell, i.e. theantigen. The term moiety comprises, e.g., an antibody or antibodyfragment. The term “antibody” as used herein refers to polyclonal ormonoclonal antibodies which can be generated by methods well known tothe person skilled in the art. The antibody may be of any species, e.g.murine, rat, sheep, human. For therapeutic purposes, if non-humanantigen binding fragments are to be used, these can be humanized by anymethod known in the art. The antibodies may also be modified antibodies(e.g. oligomers, reduced, oxidized and labelled antibodies). The term“antibody” comprises both intact molecules and antibody fragments, suchas Fab, Fab′, F(ab′)2, Fv and single-chain antibodies. Additionally, theterm “marker-binding fragment” includes any moiety other than antibodiesor antibody fragments that binds preferentially to the desired targetmolecule of the cell. Suitable moieties include, without limitation,oligonucleotides known as aptamers that bind to desired target molecules(Hermann and Pantel, 2000: Science 289: 820-825), carbohydrates, lectinsor any other antigen binding protein (e.g. receptor-ligand interaction).

“retentate” as used herein refers to the material that does not passthrough a filter, mesh or membrane.

“ultimate utility” as used herein refers to manufacture of or direct usein regenerative medicines, adoptive cell therapies, ATMPs, diagnostic invitro studies or scientific research.

With reference to FIG. 1 there is disclosed:

-   1 a Flexible container for: disaggregation; and digestion in the    embodiment involving enzymatic digestion.-   1 b Open end for transfer of solid tissue materials into container 1    a-   1 c hanging holes to support container 1 a-   1 d target heat weld location to seal container 1 a using heat    welder 13 m-   1 e rounded edges on internal container 1 a surfaces to reduce    losses which may occur as part of transfer to examples illustrated    in FIG. 2 (a, b or c) or FIG. 3 (a or b)-   1 f tubing 1 f enables media 3 a to be transferred into container 1    a via sterile filter 2 a-   1 g in example tubing 1 g enables digestion enzymes 3 b to be    transferred into container 1 a via sterile filter 2 b-   1 h after disaggregation, especially involving enzymatic digestion a    phase of incubation, the mixture is transferred out via tubing 1 h    via filter unit 4 a containing filter 4 b prior to entering-   2 a spike and sterile filter for media 3 a-   2 b spike and sterile filter for enzymes 3 b in one example, where    enzymes are required-   3 a media for disaggregation and in one example enzymatic digestion-   3 b enzymes for disaggregation in one example-   4 a flexible filter unit-   4 b non-disaggregated tissue filter-   5 a tubing clamp to allow media (3 a) to enter the flexible    container 1 a via filter 2 a-   5 b in one example where enzymes are used a tubing clamp will allow    enzymes (3 b) to enter the flexible container 1 a via filter 2 b-   5 c tubing clamp to allow contents of flexible container 1 a to pass    via filter 4 a into one or more examples identified in FIG. 2 (a-c)    Or FIG. 3 (a or b)-   FIG. 2a provides a further example of the invention in which:-   2 c spike and sterile filter for media-   3 a in one example short term storage media-   3 c freezing solution a media required for cryopreservation in one    of the examples illustrated in FIG. 2a or FIG. 3b-   4 c in one example an additional flexible filter module containing    filters 4 d & 4 e-   4 d in one example FIG. 2a a flexible filter unit may be required    for additional size segregation of cell/tissue clumps-   4 e in one example FIG. 2a a flexible filter unit is required to    retain cells but allow the media/cell fragments to be washed out-   5 d in one example FIG. 2a tubing clamp is in place to stop material    from container 1 a that has passed though 4 a & 4 c from returning    back to container 1 a-   5 e in one example FIG. 2a tubing clamp is in place to allow waste    material from container 1 a that has passed through 4 a, 4 c and 4 e    to enter container 6 a but stop media (3 a or 3 c) entering via    filter 2 c from entering container 6 a-   5 f both tubing clamps stop material from container 1 a that has    passed though filters 4 a, 4 c and 4 e from entering the tubing to    the media container (3 a or 3 c) or transferring to one of the    examples FIG. 3 (a or b) before the waste has passed into container    6 a via 5 e. Once the waste has been depleted then tubing clamps 5 e    and 5 d close and both tubing clamps 5 f allowing media (3 a or 3 c)    to transfer cells within filter 4 e into one of the examples    identified in FIG. 3 (a orb)-   6 a a waste container-   6 b hanging holes to support container 6 a-   FIG. 2b provides yet a further example in which:-   5 g a tubing clamp in place to allow contents of container 1 a to    enter the flexible container 7 a via filter 4 a-   5 h a tubing clamp in place to allow contents of container 7 a to    pass through filter 8 a retaining and enriching for cells while    allowing waste and debris to pass through filter 8 b into container    6 a with the pressure controlled by valve 8 c before the enriched    cells return to container 7 a via an open clamp 5 i-   5 i a tubing clamp is in place to allow contents of container 7 a    via open tubing clamp 5 h to pass through filter 8 a retaining and    enriching for cells while allowing waste and debris to pass through    filter 8 b into container 6 a with the pressure controlled by valve    8 c before the enriched cells return to container 7 a-   5 j after cell enrichment has occurred then tubing clamp 5 h closes    and 5 j opens allowing contents of 7 a to pass on to one of the    examples FIG. 3 (a or b)-   6 a a waste container-   6 b hanging holes to support container 6 a-   7 a a flexible container to receive the contents of: 1 a via filter    4 a; and filter 8 a-   7 b hanging holes to support container 7 a-   7 c rounded edges on internal container 7 a to reduce losses which    may occur as part of transfer to examples illustrated in FIG. 3 (a    or b)-   7 d tubing to allow container 7 a to receive the contents of: 1 a    via filter 4 a; and filter 8 a-   7 e tubing to allow contents of container 7 a to pass through filter    8 a retaining and enriching for cells while allowing waste and    debris to pass through filter 8 b into container 6 a with the    pressure controlled by valve 8 c before the enriched cells return to    container 7 a via an open clamp 5 i-   7 f tubing to allow contents of container 7 a via open tubing clamp    5 h to pass through filter 8 a retaining and enriching for cells    while allowing waste and debris to pass through filter 8 b into    container 6 a with the pressure controlled by valve 8 c before the    enriched cells return to container 7 a-   8 a contents of container 7 a can be filtered to remove waste media    and debris via filter-   8 b while enriching for cells under the control of valve 8 c before    returning to container 7 a-   8 b & 8 c see 8 a

In one example, as shown in FIG. 2c it is described that

-   5 g a tubing clamp in place to allow contents of container 1 a to    enter the flexible container 7 a via filter 4 a-   5 h a tubing clamp in place to allow contents of container 7 a to    pass through filter 9 a retaining and enriching for cells while    allowing waste and debris to pass through filter 9 b into container    6 a with the pressure controlled by valve 9 c before the enriched    cells return to container 7 a via an open clamp 5 i-   5 i a tubing clamp is in place to allow contents of container 7 a    via open tubing clamp 5 h to pass through filter 9 a retaining and    enriching for cells while allowing waste and debris to pass through    filter 9 b into container 6 a with the pressure controlled by valve    9 c before the enriched cells return to container 7 a-   5 j after cell enrichment has occurred then tubing clamp 5 h closes    and 5 j opens allowing contents of 7 a to pass on to one of the    examples FIG. 3 (a or b)-   6 a a waste container-   6 b hanging holes to support container 6 a-   7 a a flexible container to receive the contents of: 1 a via filter    4 a; and filter 9 a-   7 b hanging holes to support container 7 a-   7 c rounded edges on internal container 7 a to reduce losses which    may occur as part of transfer to examples illustrated in FIG. 3 (a    or b)-   7 d tubing to allow container 7 a to receive the contents of: 1 a    via filter 4 a; and filter 9 a-   7 e tubing to allow contents of container 7 a to pass through filter    9 a retaining and enriching for cells while allowing waste and    debris to pass through filter 9 b into container 6 a with the    pressure controlled by valve 9 c before the enriched cells return to    container 7 a via an open clamp 5 i-   7 f tubing to allow contents of container 7 a via open tubing clamp    5 h to pass through filter 9 a retaining and enriching for cells    while allowing waste and debris to pass through filter 9 b into    container 6 a with the pressure controlled by valve 9 c before the    enriched cells return to container 7 a-   9 a contents of container 7 a can be filtered to remove waste media    and debris via filter 9 b while enriching for cells under the    control of valve 9 c before returning to container 7 a-   9 b & 9 c see 9 a

FIG. 3a provides yet a further example of the invention in which:

-   5 k a tubing clamp is in place to allow the contents of: 1 a (in    example FIG. 1 via filter 4 a or in example FIG. 2a via filter 4 c);    or 7 a (in example FIG. 2b via filter 8 a or in example FIG. 2c via    filter 9 a) to be transferred into container 10 a-   10 a a flexible container to receive the contents of: 1 a via filter    4 a (in example FIG. 1) where examples described in FIG. 2 (a, b    or c) are not required; 1 a via filters 4 a & 4 c (in example FIG.    2a ); 7 a via filter 8 a (in example FIG. 2b ); or 7 a via filter 9    a (in example FIG. 2c )-   10 b hanging holes to support container 10 a-   10 c rounded edges on internal container 10 a to reduce losses which    may occur as part of transfer out via 10 e or f-   10 d tubing to enable container 10 a to receive the contents of: 1 a    via filter 4 a (in example FIG. 1) where examples described in FIG.    2 (a, b or c) are not required; 1 a via filters 4 a & 4 c (in    example FIG. 2a ); 7 a via filter 8 a (in example FIG. 2b ); or 7 a    via filter 9 a (in example FIG. 2c )-   10 e tubing to enable contents of container 10 a to be withdrawn via    connector 10 h-   10 f tubing with a flexible membrane to enable a sterile spike to be    introduced via cover-   10 g to enable contents of container 10 a to be withdrawn 10 g    aseptic cover for tubing containing membrane 10 f-   10 h connector to enable contents of 10 a to be withdrawn via tubing    10 e

In a further example, as shown in FIG. 3b there is provided:

-   2 c spike and sterile filter for media (3 c)-   3 c media required for cryopreservation-   5 l tubing clamp to allow the contents of: 1 a (in example FIG. 1    via filter 4 a or in example FIG. 2a via filter 4 c); or 7 a (in    example FIG. 2b via filter 8 a or in example FIG. 2c via filter 9 a)    to be transferred into container 11 a-   5 m tubing clamp to allow media (3 c) to enter the flexible    container 11 a via filter and spike 2 c-   5 n tubing clamp to allow contents of container 11 a to enter one of    the 12 a containers depending on the open or closed status of tubing    clamps 5 o to 5 t-   5 o-5 t tubing clamps to allow contents of container 11 a to enter    one of the 12 a containers depending on the open or closed status of    tubing clamps 5 o to 5 t-   11 a a flexible container to receive the contents of: 1 a via filter    4 a (in example FIG. 1) where examples described in FIG. 2 (a, b    or c) are not required; 1 a via filters 4 a & 4 c (in example FIG.    2a ); 7 a via filter 8 a (in example FIG. 2b ); or 7 a via filter 9    a (in example FIG. 2c )-   11 b hanging holes to support container 11 a-   11 c rounded edges on internal container 11 a to reduce losses which    may occur as part of transfer out via 11 f-   11 d tubing to enable container 10 a to receive the contents of: 1 a    via filter 4 a (in example FIG. 1) where examples described in FIG.    2 (a, b or c) are not required; 1 a via filters 4 a & 4 c (in    example FIG. 2a ); 7 a via filter 8 a (in example FIG. 2b ); or 7 a    via filter 9 a (in example FIG. 2c )-   11 e tubing to allow cryopreservation media 3 c to be transferred    into container 11 b-   11 f tubing to enable the contents of 11 a to be transferred to    container(s) 12 a-   12 a flexible containers to cryopreserve and store the final    disaggregated cells product.-   12 b a fixture allowing aseptic transfer of the cells out of the    container (12 a)-   12 c a space as part of 12 a suitable for the volume to be stored-   12 d a target location for welding the tubing and secondary flexible    container as part of 12 a using welder 13 n

FIG. 4 shows a further example of the device and kit of the invention inwhich:

-   13 a Pegs for hanging media 3 a, 3 b, 3 c-   13 b pegs connected to weight sensors for hanging containers 1 a and    depending on the examples used these could include one or more of: 7    a, 10 a & 11 a. Where the weight sensors are used to define decision    stages to control the automated processing of the materials-   13 c Heat welder to seal container 1 a at target site 1 d after    tissue has been introduced-   13 d disaggregation module with an opening that can be closed and    locked to enable disaggregation and in the example that uses digest    enzymes is capable of controlling temperatures between 0° C. and    40° C. to a tolerance of 1° C. to enable digestion. The module also    has a built in sensor to assess the level of solid tissue    disaggregation by determining the variation in light distribution    against time to identify change and thereby identifying completion    of the disaggregation process which will occur over a period of    seconds to hours.-   13 e final formulation module with an enclosure to allow temperature    control of either container 10 a or 11 a depending on the example    used which is capable of controlling temperatures between 0° C. and    ambient environmental temperature to a tolerance of 1° C.-   13 f disaggregation surfaces which come directly into contact with    container 1 a and pushes against the back of the module 13 d    enclosure which can be closed and locked during disaggregation and    digestion where enzymes are utilised.-   13 g tubing clamp-   13 j tubing clamp-   13 h peristaltic tubing pumps-   13 i tubing locators-   13 k tubing valve required for examples FIGS. 2b & 2 c-   13 l Pegs for hanging containers depending on the examples used    these could include one or more of: 6 a & 12 a-   13 m tubing welder and cutter required for example FIG. 3b for    tubing to container(s) 12 a-   13 n tubing welder required for example FIG. 3b for tubing to    container(s) 12 a at target location 12 d-   13 o controlled rate cooling module capable of cooling or    maintaining any temperature between 8° C. and at least −80° C.

Example Method

The method of the invention is exemplified according to the followingprocess. It is clearly stated that other than the essential features ofthe method, the various optional steps listed herein can beindependently combined to achieve the relevant technical advantagesassociated with the type of sampling and result to be achieved.

A semi-automatic aseptic tissue processing method comprising:automatically determining aseptic disaggregation tissue processing stepsand one or more further tissue processing steps and their associatedconditions from a digital tag identifier on an aseptic processing kit,optionally in accordance with the kit described herein; placing a tissuesample into a flexible plastic container of the aseptic processing kit;and processing the tissue sample by automatically executing the one ormore tissue processing steps by communicating with and controlling thedisaggregation module; the optional enrichment module; and thestabilisation module.

Essentially the process may comprise taking an open ended bag (1stflexible container that is part of disaggregation module) that willreceive the biopsy/tissue sample which is already connected via one ormore conduits to (conduit) or can be connected via a manual operatorcontrolled aseptic connection to

I. a single container with digestion media (2nd flexible container thatis part of the disaggregation module) and with or without astabilisation solution (same 2nd flexible container is part of thestabilisation module also)

II. one container with a digestion solution (2nd flexible container thatis part of the disaggregation module) & another container with astabilisation solution (4th flexible container is part of thestabilisation module)

on addition of the biopsy and sealing of the open ended bag thedigestion media can be added via the conduit or aseptic connections(conduit/ports claim 1) and the tissue material processed.

On completion of the digestion by which point the tissue is now a singleor small number aggregate cellular suspension the cells can optionallybe filtered prior to step 4 (optional enrichment module for filtrationcomprises the 1st flexible container containing sample and filtered to a3rd container for receiving the enriched filtrate)

Where the stabilisation media is not present in the same flexiblecontainer i.e. option 2.II.

this will require the container with stabilisation solution to be addedby opening the attached conduit or manual operator controlledaseptically connection to be competed and said connection to be openedenabling in both cases the stabilisation solution to be added before theprocess continues.

The single or small number aggregate cellular suspension in the originalflexible container or which may be optionally subdivided into multiplestorage stabilisation containers thereafter are maintained in a stablestate on the device and/or will undergo cryopreservation prior toremoval for, transport, storage and or used in their ultimately utility.(The stabilisation module also comprises 1st or 3rd container as used instorage/freezing/storage)

In one further non-limiting example of the process:

-   a) Collection of tissue sample by a separate procedure such as    biopsy's or surgery to collect the required tissue material (not    part of the invention) is placed into the initial flexible plastic    container (see FIG. 1-container 1 a).-   b) Media (see example FIG. 1-media 3 a) is transferred into the    disaggregation chamber, or in one example also enters and collects    enzymes (see FIG. 1-enzymes 3 b), prior to disaggregation using one    or more of the following examples of the invention a mechanism such    as weight sensors (see FIGS. 1-13 b as part of module 13 d) will    assess the required amount of media to add either determined by:    direct operator input or weight of solid tissue.-   c) The single use flexible disaggregation container, solid tissue,    media and in one example enzymes are combined during a physical    disaggregation process for a minimum of a few seconds up to several    hours with an optimal time of between 1 and 10 minutes required to    break up the solid tissue until there is no visual change (FIG. 5    including Table 1). The disaggregation device is designed to    compress the tissues using a variable speed and time depending upon    the time taken to disaggregate and feedback via sensors within the    disaggregation module (see example FIG. 1-13 d).-   d) In one embodiment where enzymes are present this will require    incubation periods at an optimal temperature of between 30 & 37° C.    but could be as low as 0° C. up to 40° C. for at least 1 minute to    several hours but more preferable 15 to 45 minutes.-   e) Step c and in the embodiment where enzymes step d) can be    repeated until the tissue stops changing or the see example has been    disaggregated into a liquid cell suspension whichever comes 1st    monitored by a sensor in the disaggregation module disaggregation    module (FIG. 1-13 d).-   f) In one embodiment incompletely disaggregated tissues, associated    material and impurities are removed enabling enrichment of the cell    suspension by passing the disaggregated tissue and media using one    or more of the following embodiments:-   i. Direct pass through one or more mechanical filters with holes at    least >0.1 μm to 1000 μm but most preferably between 50 and 250 μm    and more preferably 100 μm to 200 μm (illustrated in FIG. 2a )-   ii. Density based separation using centrifugation and/or    sedimentation with or without a cell aligned density retention    solution (e.g. Ficoll-paque GE Healthcare).-   iii. Hydrodynamic filtration where fluid flow and flow obstructing    materials enhance the resolution and fractionation of the cells and    impurities based on size and shape-   iv. Field flow fractionation where an applied field (e.g. flow,    electric, gravitational, centrifugal) acts in a perpendicular or    reverse direction to the selection flow (e.g. Tangential flow    filtration, Hollow fibre flow filtration, Asymmetric flow    filtration, Centrifugal flow filtration). In which case: cells or    impurities which are most responsive to the force are driven to the    wall where flow is lowest and therefore a long retention time; while    cells or impurities which are least responsive to the force remain    laminar to the flow and elute quickly (tangential flow filtration    illustrated in FIG. 2b & c)-   v. Acoustophoresis where one or more an acoustic frequency(ies)    tuned to or harmonized with populations of cells or impurities is    used to drive the required cells or impurities in a tangential path    to the input stream.-   g) In one embodiment the disaggregated enriched tissue product will    be resuspended in a fresh media (FIG. 2a using media 3 a) such as:-   i. a cell enrichment media in order to undergo an independent    targeted enrichment procedure as described previously-   ii. direct cell culture or cold storage media (such as    HypoThermosol® from BioLife Solutions.-   h) in the embodiment employed in g) the resuspended disaggregated    solid tissue derived product will be transferred to one of the    embodiment final product containers (illustrated in FIG. 3a ) for    storage for hours to days prior to being used for its ultimate    utility.-   i) otherwise after step f) the embodiment (illustrated in FIG. 3b )    will apply where the disaggregated solid tissue derived product will    undergo re-suspension in a cryoprotectant (FIG. 3b -media 3 c) a    freezing solution for storage of the disaggregated solid tissue    derived product for days to years such as CryoStor® Freezing    solution from BioLife Solution.-   j) At this stage the disaggregated solid tissue derived product    re-suspended in freezing solution using the device (FIG. 4-module 13    e) will be transferred to 1 or more flexible cryopreservation    container(s) (illustrated in FIG. 3a -container 12 a) and in one    embodiment of the device it will perform a controlled rate freezing    process using the device (FIG. 4-module 13 o).-   k) After which the bags can be separated from the device and aseptic    processing kit for independent storage or distribution.

FIGS. 6 and 7 describe further examples in which the disposable kit ofthe invention can be used with an automatic device for semi-automaticaseptic processing of tissue samples.

FIG. 6 describes the following semi-automatic aseptic tissue processingmethod using multiple flexible containers for different startingsolutions that are part of the modules of the process used fordisaggregation and stabilisation.

Process step 1—The user may login to device and scan the tag on theaseptic kit using the device to transfer the automatic processing stepsto be used. The device processor recognises the tag and is provided withinformation needed to carry out the specific processing instructionsrelated to that particular kit.

Process step 2—The digestion media containing flexible bag (part ofdisaggregation module) and cryo/stabilisation solution containingflexible bag (part of the stabilisation module) are each hung or securedto the device.

Process step 3—The biopsy or tissue sample for processing may be placedinto a flexible container (part of both modules) of the aseptic kit viaan open end.

Process step 4—The flexible container comprising the sample may then besealed using a heat weld to close the open end (used to add the sampleduring initial processing).

Process step 5—The user may then interact with the user interface of theprocessor to confirm the tissue sample is present and enter any furthertissue material specific information, if required.

Process step 6—Digestion media and cryo/stabilisation solution flexiblecontainers are connected with the flexible container housing the sample,after which it maybe placed into the device for automatic processing.

Process step 7—The device executes the cycles according to the kitinformation undertaking disaggregation of the sample andstabilisation/cryo preservation of resulting cells.

Process step 8—When stabilised/frozen disconnect and discard used mediaand cryo/stabilisation containers of kit. Tissue processed into singleor multi-cell solution in flexible container is disconnected beforetransferring into storage or transport container prior to its ultimateutilisation.

FIG. 7 describes how flexible containers comprising the media used inthe process may be shared between the modules of the aseptic processingkit and method.

Process step 1—The user may login to device and scan the tag on theaseptic kit using the device to transfer the automatic processing stepsto be used.

Process step 2—A flexible bag (part of disaggregation/stabilisationmodule) comprising both the media and cryo/stabilisation solution ishung or otherwise secured to the device.

Process step 3—The biopsy or tissue sample for processing may be placedinto a further flexible container (part of both modules) of the aseptickit via an open end.

Process step 4—The flexible container comprising the sample may then besealed using a heat weld to close the open end.

Process step 5—The user may then interact with the user interface of theprocessor to confirm the tissue sample is present and enter any tissuematerial specific information, if required.

Process step 6—Digestion media and cryo/stabilisation solution flexiblecontainer is connected with the flexible container housing the sample,after which it maybe placed into the device for automatic processing.

Process step 7—The device cycles to enable disaggregation of the sampleand stabilisation of resulting cells, optionally via cryopreservation.

Process step 8—When freezing/stabilising is complete the userdisconnects and discard used flexible containers of kit. Tissueprocessed into single or multi-cell solution in the remaining flexiblecontainer is disconnected before transferring into storage or transportcontainer prior to its ultimate utilisation.

Enzymatic Digestion

By way of example, in another embodiment of the method of the invention,where the disaggregation process is being supplemented with enzymaticdigestion the media formulation for enzymatic digestion must besupplemented with enzymes that aid in protein breakdown causing the cellto cell boundaries to breakdown as described above.

Media Formulation for Enzymatic Digestion

Various liquid formulations known in the art of cell culturing or cellhandling can be used as the liquid formulation used for celldisaggregation and enzymatic digestion of solid tissues, including butnot limited to one or more of the following media Organ PreservationSolutions, selective lysis solutions, PBS, DMEM, HBSS, DPBS, RPMI,Iscove's medium, X-VIVO™, AIM-V™, Lactated Ringer's solution, Ringer'sacetate, saline, PLASMALYTE™ solution, crystalloid solutions and IVfluids, colloid solutions and IV fluids, five percent dextrose in water(D5W), Hartmann's SolutionDMEM, HBSS, DPBS, RPMI, AIM-V™, Iscove'smedium, X-VIVO™, each can be optionally supplemented with additionalcell supporting factors e.g. with foetal calf serum, human serum orserum substitutes or other nutrients or Cytokines to aid in cellrecovery and survival or specific cell depletion. The media can bestandard cell media like the above mentioned media or special media fore.g. primary human cell culture (e.g. for endothelia cells, hepatocytesor keratinocytes) or stem cells (e.g. dendritic cell maturation,hematopoietic expansion, keratonocytes, mesenchymal stem cells or Tcells). The media may have supplements or reagents well known in theart, e.g. albumins and transport proteins, amino acids and vitamins,metal-ion(s), antibiotics, attachments factors, de-attachment factors,surfactants, growth factors and cytokines, hormones or solubilisingagents. Various media are commercially available e. g. fromThermoFisher, Lonza or Sigma-Aldrich or similar media manufacturers andsuppliers.

The liquid formulation required for enzymatic digestion must havesufficient calcium ions present in the of at least 0.1 mM up to 50 mMwith an optimal range of 2 to 7 mM ideally 5 mM.

The solid tissue to be digested can be washed after disaggregation witha liquid formulation containing chelating agents EGTA and EDTA to removeadhesion factors and inhibitory proteins prior to washing and removal ofEDTA and EGTA prior to enzymatic digestion.

The liquid formulation required for enzymatic digestion is more optimalwith minimal chelating agents EGTA and EDTA which can severely inhibitenzyme activity by removing calcium ions required for enzyme stabilityand activity. In addition β-mercaptoethanol, cysteine and8-hydroxyquinoline-5-sulfonate are other known inhibitory substances.

Cryopreservation

As described in preferred embodiments final cell container forcryopreservation is a flexible container manufactured from resilientdeformable material. In this embodiment of the device the finalcontainer is either transferred directly to a freezer −20 to −190° C. ormore optimally located in the controlled rate freezing apparatus eitherassociated with the device or supplied separately (manufactured by forexample Planer Products or Asymptote Ltd) in which the temperature ofthe freezing chamber and the flexible storage container(s) employed tocontain the enriched disaggregated solid tissue container is controlledeither by: injecting a cold gas (normally nitrogen for example Planerproducts); or by removing heat away from the controlled coolingsurface(s). Both methods result in the ability to accurately controlwith an error of less than 1° C. or more preferable 0.1° C. the freezingprocess at the required rate for the specific cell(s) to be frozen basedon the freezing solution and the desired viability of the product. Thiscryopreservation process must take into account the ice nucleationtemperature which is ideally as close as possible to the meltingtemperature of the freezing solution. Followed by crystal growth in anaqueous solution, water is removed from the system as ice, and theconcentration of the residual unfrozen solution increases. As thetemperature is lowered, more ice forms, decreasing the residualnon-frozen fraction which further increases in concentration. In aqueoussolutions, there exists a large temperature range in which ice co-existswith a concentrated aqueous solution. Eventually through temperaturereduction the solution reaches the glass transition state at which pointthe freezing solution and cells move from a viscous solution to asolid-like state below this temperature the cells can undergo no furtherbiological changes and hence are stabilised, for years potentiallydecades, until required.

Further Applications of the Invention

The disaggregated cell products achieved by the method of the presentinvention can be cultured and/or analysed (characterised) according toall methods known to the person skilled in the art.

The cells obtainable by the methods disclosed herein may be used forsubsequent steps such as research, diagnostics, tissue-banks, biobanks,pharmacological or clinical applications known to the person skilled inthe art. Cells can then be taken into culture using a Medium optimizedfor this application, e.g. T cell Mixed Media (Cellular Therapeutics)usually containing but not limited to growth factors such as IL-2, IL-7,IL-15, IL-21 or stimulatory conditions such as plates or polystyrenebeads coated with antibodies. In the present invention isolated cellswere seeded into culture containers and maintained using proceduresstandardly used by a person skilled in the art such as a humidifiedatmosphere (1-20% usually 5% CO2, 80 to 99% usually 95% air) attemperatures between 1 to 40 usually 37° C. for several weeks andsupplements may be added supplemented with 10% FBS and 3000 IU/mL IL-2.

Such cell cultures can be used to study e.g. cell function, tumour cellkilling, cell signalling, biomarkers, cell pathways, nucleic acids, andother cell or tissue related factors that may be used to identify donor,tissue, cell or nucleic acid status.

The enriched cells could be used before and/or after cell culturing as apharmaceutical composition in the therapy, e.g. cellular therapy, orprevention of diseases. The pharmaceutical composition can be used forthe treatment and/or prevention of diseases in mammals, especiallyhumans, possibly including administration of a pharmaceuticallyeffective amount of the pharmaceutical composition to the mammal.

The disease may be any disease, which can be treated and/or preventedthrough the presence of solid tissue derived cells and/or throughincreasing the concentration of the relevant cells in/at the relevantplace, i.e. the tumours or sites of disease. The treated and/orpreventively treated disease may be any disorder, e.g. cancer or adegenerative disorder. The treatment may be the transplantation ofenriched, engineered or expanded cells or any combination of these andeither administered to the relevant part of the body or suppliedsystemically.

Pharmaceutical compositions of the present disclosure may beadministered in a manner appropriate to the disease to be treated (orprevented). The quantity and frequency of administration will bedetermined by such factors as the condition of the patient, and the typeand severity of the patient's disease, although appropriate dosages maybe determined by clinical trials.

Further specific examples:

Example 1

Impact of the Length of Disaggregation

Peripheral blood mononuclear cells were physically disaggregated for 0,1, 5 & 10 minutes continuously before a being cultured in vitro for 0,24 & 96 hours to assess cell recovery. The results demonstrate thephysical process has negligible impact over 1 or 5 minutes and at 10minutes the impact was transient where and initial reduction in viablecells at 0 hours was equivalent to non-disaggregated cells at 24 & 96hours (FIG. 5).

Example 2

Solid Tissue Sample Size, Volume of Digestion Media, Disaggregation andIncubation Times

Conditions of: Solid tissue size, volume of digestion media,disaggregation time and incubation conditions have been tested anddemonstrate full disaggregation of solid tissue (Table 1) except wherethe volume of digestion media cushioned the solid tissue during thedisaggregation process resulting in 30-50% of the solid tissue remainingintact.

EQUIVALENTS

The details of one or more embodiments of the disclosure are set forthin the accompanying description above. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, the preferred methodsand materials are now described. Other features, objects, and advantagesof the disclosure will be apparent from the description and from theclaims. In the specification and the appended claims, the singular formsinclude plural referents unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

The foregoing description has been presented only for the purposes ofillustration and is not intended to limit the disclosure to the preciseform disclosed, but by the claims appended hereto.

NON-PATENT LITERATURE CITED

-   Miller R G and Phillips R A. Separation of cells by velocity    sedimentation. J Cell Physiol 1969; 73: 191-201-   Buckner D, Graw R G, Eisel R J, et al. Leukapheresis by continuous    flow centrifugation (CFC) in patients with chronic myelocytic    leukemia (CML). Blood 1969; 33: 353-369-   Liu W, Hou Y, Chen H, et al. Sample preparation method for isolation    of single-cell types from mouse liver for proteomic studies.    Proteomics 2011; 11: 3556-3564-   Nagase K, Kimura A, Shimizu T, et al. Dynamically cell separating    thermo-functional biointerfaces with densely packed polymer brushes.    J Mater Chem 2012; 22: 19514-19522-   Rembaum A, Yen R C K, Kempner D H, et al. Cell labelling and    magnetic separation by means of immunoreagents based on polyacrolein    microspheres. J Immunol Methods 1982; 52: 341-351.-   Cahoy J D, Emery B, Kaushal A, et al. A transcriptome database for    astrocytes, neurons, and oligodendrocytes: a new resource for    understanding brain development and function. J Neurosci 2008;    28:264-278-   Miltenyi S, Muller W, Weichel W, et al. High gradient magnetic cell    separation with MACS. Cytometry 1990; 11:231-238.-   Topalian S L, Muul L M, Solomon D, et al. Expansion of human tumor    infiltrating lymphocytes for use in immunotherapy trials. J Immunol    Methods. 1987; 102(1):127-41.-   Bonner W A, Sweet R G, Hulett H R, et al. Fluorescence activated    cell sorting. Rev Sci lnstrum 1972; 43: 404-409-   Gossett D R, Weaver W M, Mach A J. Et al., Label-free cell    separation and sorting in microfluidic systems, Anal Bioanal Chem,    2010, 397, 3249-3267-   Barbara Cunha B, Peixoto C, Silva M M, et al., Filtration    methodologies for the clarification and concentration of human    mesenchymal stem cells, J. of Membrane Sci., 2015, 478, 117-129-   Klein A B, Witonsky S G, Ahmed S A, et al. Impact of different cell    isolation techniques on lymphocyte viability and function. J    Immunoassay Immunochem 2006; 27: 61-76-   Steinberg M S. ‘ECM’: its nature, origin and function in cell    aggregation. Exp Cell Res 1963; 30: 257-279.-   Hefeneider S H, McCoy S L, Morton J I, et al. DNA binding to mouse    cells is mediated by cell-surface molecules: the role of these    DNA-binding molecules as target antigens in murine lupus. Lupus    1992; 1: 167-173.-   Pisetsky D S and Fairhurst A-M. The origin of extracellular DNA    during the clearance of dead and dying cells—review. Autoimmunity    2007; 40: 281-284-   Renner W A, Jordan M, Eppenberger H M, et al. Cell-cell adhesion and    aggregation: influence on the growth behaviour of CHO cells.    Biotechnol Bioeng 1993; 41: 188-193-   Shedlock D J, Aviles J, Talbott K T et al., Induction of Broad    Cytotoxic T Cells by Protective DNA Vaccination Against Marburg and    Ebola. Molecular Therapy, 2013; 21, 1432-1444-   Baust J G, & Baust J M, Advances in Biopreservation, 2006, Chapt. 8,    157-196-   Seglen, P. O., Preparation of Isolated Rat Liver Cells, Methods in    Cell Biology, 1976; 13, 29-   Quistorff, B., Dich, J., & Grunnet, N. Preparation of isolated rat    liver hepatocytes. Methods in molecular biology, Chapt 14, 1990;    151-160.-   Seifter, S., Gallop, P. M., Klein, L., et al. Studies on Collagen,    Part II. Properties of Purified Collagenase and Its Inhibition. J.    Biol. Chem. 1959; 234:285

1: A single use aseptic kit comprising: a disaggregation module forreceipt and processing of material comprising solid mammalian tissue;and a stabilisation module for further processing and/or storingdisaggregated product material, wherein each of said modules comprisesone or more flexible containers connected by one or more conduitsadapted to enable flow of the tissue material there between, and whereineach of said modules comprises one or more ports to permit aseptic inputof media and/or reagents into the one or more flexible containers,wherein the kit further comprises a digital, electronic, orelectromagnetic tag identifier relating to a specific program thatdefines a type of disaggregation, enrichment, and/or stabilisationprocess and use of one or more types of media in the process(es). 2: Thesingle use aseptic kit of claim 1, wherein the one or more flexiblecontainers comprise a resilient deformable material. 3: The single useaseptic kit of claim 1, wherein the one or more flexible containers ofthe disaggregation module comprises one or more sealable openings. 4:The single use aseptic kit of claim 3, wherein the flexible container ofthe disaggregation module comprises a heat sealable weld. 5: The singleuse aseptic kit of claim 1, wherein the one or more flexible containerscomprises internally rounded edges. 6: The single use aseptic kit ofclaim 1, wherein the one or more flexible containers of thedisaggregation module comprises disaggregation surfaces adapted tomechanically crush and shear the solid tissue therein. 7: The single useaseptic kit of claim 1, further comprising an enrichment modulecomprising one or more flexible containers, wherein the enrichmentmodule is configured for filtration of disaggregated solid tissuematerial and segregation of non-disaggregated tissue and filtrate andcomprising, wherein the enrichment module is connected to thestabilisation and desegregation modules by one or more conduits adaptedto enable flow of the tissue material, and wherein the one or moreflexible containers of the enrichment module each comprise a filter thatis configured to retain a retentate of cellularised disaggregated solidtissue. 8: The single use aseptic kit of claim 1, wherein the one ormore flexible containers of the stabilisation module comprises mediaformulation for storage of viable cells in solution or in acryopreserved state.
 9. (canceled)
 10. (canceled) 11: The single useaseptic kit of claim 7, wherein the same flexible container forms partof one or more of the disaggregation modules, stabilisation modules,and/or enrichment modules. 12: The single use aseptic kit of claim 11,wherein the disaggregation module comprises a first flexible containerfor receipt of the tissue to be processed. 13: The single use aseptickit of claim 12, wherein the disaggregation module comprises a secondflexible container comprising the media for disaggregation. 14: Thesingle use aseptic kit of claim 13, wherein the enrichment modulecomprises a first flexible container and a third flexible containerconfigured to receive enriched filtrate. 15: The single use aseptic kitof claim 14, wherein both the disaggregation module and thestabilisation module comprise a second flexible container and whereinthe second container comprises digestion media and stabilisation media.16: The single use aseptic kit of claim 15, wherein the stabilisationmodule comprises a fourth flexible container comprising stabilisationmedia. 17: The single use aseptic kit of claim 16, wherein thestabilisation module also comprises the first flexible container and/orthe third flexible container for storing and/or undergoingcryopreservation. 18: Use of the single use aseptic kit according toclaim 1 in a semi-automated process for the aseptic disaggregation,stabilisation, and enrichment of mammalian cells or cell aggregates. 19:An automated device for semi-automated aseptic disaggregation,enrichment, and/or stabilisation of cells or cell aggregates frommammalian solid tissue comprising: a programmable processor; and aradio-frequency identification tag reader, wherein the programmableprocessor is configured to recognise a single-use aseptic kit inaccordance with claim 1 using the tag reader and is adapted tosubsequently execute a kit-specific program, wherein the program definesthe type of disaggregation, enrichment, and/or stabilisation processesand the media type(s) required for use in the process(es). 20-21.(canceled) 22: The automated device of claim 19, wherein theprogrammable processor is adapted to communicate with and control one ormore of: the disaggregation module; the enrichment module; and thestabilisation module. 23: The automated device of claim 22, wherein theprogrammable processor controls the disaggregation module to enable aphysical and/or biological breakdown of the solid tissue material. 24:The automated device of claim 23, wherein the programmable processorcontrols the disaggregation module to enable a physical and enzymaticbreakdown of the solid tissue material. 25: The automated device ofclaim 24, wherein the enzymatic breakdown of the solid tissue materialis by one or more media enzyme solutions selected from collagenase,trypsin, lipase, hyaluronidase, deoxyribonuclease, Liberase H1, pepsin,or mixtures thereof. 26: The automated device of claim 19, wherein theprogrammable processor controls disaggregation surfaces within thedisaggregation flexible containers that mechanically crush and shear thesolid tissue and wherein the disaggregation surfaces are mechanicalpistons. 27: The automated device of claim 19, wherein the programmableprocessor controls the stabilisation module to cryopreserve the enricheddisaggregated solid tissue in the container using a programmabletemperature. 28: The automated device of claim 19 wherein the devicefurther comprises one or more of the additional components in anycombination: sensors capable of recognising whether a disaggregationprocess has been completed in the disaggregation module prior totransfer of the disaggregated solid tissue to the enrichment module;weight sensors configured to determine an amount of media required inthe containers of one or more of the disaggregation module; theenrichment module; and/or the stabilisation module and to control thetransfer of material between respective containers; sensors to sensetemperature within the containers of the one or more of thedisaggregation module; the enrichment module; and/or the stabilisationmodule; at least one bubble sensor to control the transfer of mediabetween the input and output ports of each container in the module; atleast one peristaltic pump to control the transfer of media between theinput and output ports; pressure sensors to assess the pressure withinthe enrichment module; one or more valves to control a tangential flowfiltration process within the enrichment module; and/or one or moreclamps to control the transfer of media between the input and outputports of each module. 29: The automated device of claim 19, wherein theprogrammable processor is adapted to maintain an optimal storagetemperature range in the stabilisation module until the container isremoved; or executes a controlled freezing step. 30: The automateddevice of claim 19, further comprising a user interface. 31: Theautomated device of claim 23, wherein the interface comprises a displayscreen to display instructions that guide a user to input parameters,confirm pre-programmed steps, warn of errors, or combinations thereof.32: The automated device of claim 19, wherein the automated device isadapted to be transportable. 33: A semi-automatic aseptic tissueprocessing method comprising: automatically determining asepticdisaggregation tissue processing steps and their associated conditionsfrom a digital, electronic, or electromagnetic tag identifier associatedwith the aseptic processing kit of claim 1; placing a tissue sample intoa flexible plastic container of the disaggregation module of the asepticprocessing kit; and processing the tissue sample by automaticallyexecuting the one or more tissue processing steps by communicating withand controlling the disaggregation module; the optional enrichmentmodule; and the stabilisation module. 34: The single use aseptic kit ofclaim 1, further comprising an enrichment module for filtration ofdisaggregated solid tissue material and segregation of non-disaggregatedtissue and filtrate. 35: The single use aseptic kit of claim 1, whereinthe specific program defines use of a freezing solution for controlledrate freezing in the stabilisation module.